Local Immune Responses to Chlamydia pneumoniae in the Lungs of BALB/c Mice during Primary Infection and Reinfection

Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with Chlamydia pneumoniae. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-γ). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220⁺). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-γ) CMI response. These results suggest that repeated infections with C. pneumoniae may induce Th1-type responses with similar associated tissue reactions, as shown in C. trachomatis infection models.     

Production of Chlamydia pneumoniae proteins in Bacillus subtilis and their use in characterizing immune responses in the experimental infection model

Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the alpha-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.     

Experimental infection of Chlamydia pneumoniae in mice

NIH/S, Swiss Webster, and BALB/c mice were infected intranasally with three Chlamydia pneumoniae isolates, Kajaani 6, Helsinki 12, and TW-183. C. pneumoniae could be isolated from the lung homogenates and bronchoalveolar lavage fluids up to the third week post-infection. Specific serum IgG antibodies against C. pneumoniae reached high levels in the third week and remained elevated until the end of the 6-week follow-up period. Serum IgM levels were highest in the third week post-infection and started to decrease thereafter. In spite of these signs of ongoing infection, the mice did not show any symptoms of disease. NIH/S mice could be readily and uniformly infected, while BALB/c mice were the most resistant and developed the weakest antibody response. The greatest histological changes were detected in NIH/S mice as well. The inflammatory infiltrate, which consisted of lymphocytes and plasma cells throughout the study, was restricted to the peribronchial and perivascular space and to the interstitium of the lung parenchyma.     

Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.     

Synthetic Peptide Antisera: Their Production and Use in the Cloning of Matrix Proteins

Small amounts (0.1-1.0 nmole) of purified (>90%) proteins isolated from small quantities of connective tissues can readily be microsequenced and 20 to 40 N-terminal amino acids determined. A synthetic peptide (10 or more amino acids long) can be produced and either injected directly into rabbits for antiserum production (generally peptides >20 amino acids in length) or conjugated to a carrier protein prior to injection. The antisera have proven useful in the isolation of cDNA clones, for both locating clones producing the protein in expression libraries and in the subsequent confirmation of the sequence (preferably using a part of the sequence not directly used in the production of the original antiserum).     

A Single B-Repeat of Staphylococcus epidermidis Accumulation-Associated Protein Induces Protective Immune Responses in an Experimental Biomaterial-Associated Infection Mouse Model

Nosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device. Staphylococcus epidermidis is a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature. S. epidermidis antibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein of S. epidermidis, is considered one of the most important proteins involved in the formation of S. epidermidis biofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) of S. epidermidis RP62A Aap was developed, and the vaccine''s efficacy was evaluated in vitro with a biofilm inhibition assay and in vivo in a murine model of biomaterial-associated infection. A high IgG antibody response against S. epidermidis RP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund''s adjuvant. Sera from Brpt1.0-immunized mice inhibited in vitro S. epidermidis RP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 10⁶ CFU of S. epidermidis RP62A. Weight changes, inflammatory markers, and histological assay results after challenge with S. epidermidis indicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance to S. epidermidis RP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova)-immunized mice.     

Characterization of Clinical and Immune Responses in an Experimental Chronic Autoimmune Uveitis Model

Autoimmune uveitis is a sight-threatening intraocular inflammatory disease. For >30 years, the mouse model of experimental autoimmune uveitis has been employed to investigate disease mechanisms and test immunotherapeutic approaches. However, inflammation in this model is self-limited, and does not replicate the chronic, insidious nature prevalent in the human disease. Herein, a robust and reliable model of chronic autoimmune uveitis was developed and characterized in two strains of wild-type mice by modifying interphotoreceptor retinoid-binding protein dose and peptide fragments from conventional experimental autoimmune uveitis models. In both of these murine strains, immunization with our modified protocols resulted in a slowly progressive uveitis, with retinal scars and atrophy observed in the chronic stage by fundoscopy. Optical coherence tomography demonstrated decreased retinal thickness in chronic autoimmune uveitis mice, and electroretinography showed significantly reduced amplitudes of dark-adapted a- and b-waves and light-adapted b-waves. Histologic examination revealed prominent choroiditis with extensive retinal damage. Flow cytometry analysis showed substantially increased numbers of CD44^hiIL-17⁺IFN-γ⁻ memory T-helper 17 (Th17) cells in the retina, cervical lymph nodes, inguinal lymph nodes, and spleen. These data establish new modified protocols for inducing chronic uveitis in wild-type mice, and demonstrate a predominant memory Th17 cell response, suggesting an important role for memory Th17 cells in driving chronic inflammation in autoimmune uveitis.

Uveitis is a sight-threatening inflammatory condition of the uveal tract of the eye, comprising the iris, ciliary body, and choroid.¹ Responsible for an estimated 30,000 new cases of legal blindness each year in the United States, uveitis and its complications cause approximately 10% of irreversible vision loss.2, 3, 4, 5 The socioeconomic impact of uveitis is notable because as blindness frequently occurs in individuals of working age.⁶ In contrast to acute uveitis, which often resolves after a short course of topical corticosteroids, chronic uveitis is characterized by active inflammation that persists for at least several months, and frequently years. The persistent inflammation and its long-term requirement for corticosteroids lead to a high incidence of complications that endanger vision, including glaucoma, cataract, optic disc atrophy, and chorioretinal scars.⁷Experimental animal models have been used to recapitulate the clinical phenotype of uveitis, and have led to significant advances in our understanding of the cellular and molecular mechanisms that cause autoimmune uveitis.⁸ For example, these studies have established the critical roles of pathogenic effector T-helper 1 (Th1) and T-helper 17 (Th17) cells in the immunopathogenesis of uveitis.8, 9, 10 However, most preclinical research on autoimmune uveitis has investigated acute disease, but not the development and maintenance of chronic autoimmune uveitis (CAU).¹¹ This is because, in the widely used mouse experimental autoimmune uveitis (EAU) model, disease peaks in the first 2 to 3 weeks after immunization before resolving spontaneously.12, 13, 14, 15 Yet, as discussed, it is CAU that is associated with the greatest morbidity and risk of sight-threatening complications in the clinic, underscoring a pressing need for the development of a chronic uveitis animal model.⁷In the present study, a reliable murine model of chronic experimental autoimmune uveitis was established and characterized in two wild-type mouse strains, which exhibit clinical features consistent with noninfectious uveitis observed in humans. Furthermore, in contrast to early acute uveitis in which the immune response is characterized by both effector Th1 and effector Th17 cells, we demonstrate that CAU is primarily characterized by a robust memory Th17 response in both the retina and peripheral lymphoid compartments.     

Analysis of the Humoral Immune Response to Chlamydia pneumoniae by Immunoblotting and Immunoprecipitation

Chlamydia pneumoniae is a widely spread agent of respiratory tract infections in humans. A reliable serodiagnosis of the disease is hampered by the poor knowledge about immunodominant antigens in C. pneumoniae infections. We applied a novel strategy to identify immunogenic proteins of C. pneumoniae TW183 combining metabolic radiolabeling of de novo-synthesized chlamydial antigens with immunoprecipitation. By this technique C. pneumoniae antigens of approximately 160, 97 to 99, 60 to 62, 40, 27, and 15 kDa were detected in the vast majority of sera from patients with a current C. pneumoniae infection. By immunoblotting purified elementary bodies of C. pneumoniae TW183 with the same sera, only the 60- to 62-kDa antigen could be detected consistently. Sequential immunoprecipitation performed at different stages of the chlamydial developmental cycle revealed that the 60- to 62-kDa antigen is strongly upregulated after 24 to 48 h of host cell infection and is presented as a major immunogen in both C. pneumoniae-infected patients and mice. We conclude that, due to its high sensitivity and concurrent preservation of conformational epitopes, metabolic radiolabeling of chlamydial antigens combined with immunoprecipitation may be a useful method to reveal important immunogens in respiratory C. pneumoniae infection which might have been missed by immunoblot analysis.     

Effects of Endometrial Serpin-Like Proteins on Immune Responses in Sheep

PROBLEM: The uterine milk proteins (UTMP) are a pair of related glycoproteins that are the major secretory products of the endometrium of the pregnant ewe. UTMP are members of the serpin superfamily of serine protease inhibitors but have no known antiprotease activity. One possible role for UTMP is to inhibit uterine immune responses—UTMP inhibit mitogen and mixed lymphocyte-induced proliferation of peripheral blood lymphocytes, natural killer (NK) cell activity and abortion caused by NK cell activation. Present objectives were to further evaluate the lymphocyte-inhibitory activity of UTMP and test whether UTMP modify immune responses in vivo. METHOD: One experiment demonstrated that UTMP inhibited antigen-induced lymphocyte proliferation induced by Candida albicans extract. In another experiment, ewes were immunized with OVA mixed with 3.75 mg/ml of UTMP or ovine serum albumin (OSA control). Injections of 1 mg OVA + UTMP or OSA in incomplete adjuvant were administered 6 wk later. Titers of antibody to OVA were lower (P<0.001) for ewes administered UTMP than for ewes administered OSA. Effects of UTMP on delayed hypersensitivity reactions were evaluated in three experiments using skin-fold thickness assays. RESULTS: UTMP did not inhibit the increase in skin-fold thickness caused by PHA and Mycobacterium tuberculosis but rather tended to increase the response to PHA. CONCLUSION: Results strengthen the thesis that UTMP are physiologically relevant immunoregulatory molecules. Nonetheless, effects on skin-fold responses indicate that actions of UTMP can be more complex than would be predicted based on the proteins only having a single biological effect.     

Incidence of Chlamydia pneumoniae Infection in Vertically HIV-1 Infected Children

 The rate of seroconversion for antibody to Chlamydia pneumoniae was analysed in blood samples of 26 vertically HIV-1 infected children and 14 seroreverter children (HIV-negative children born to HIV-positive mothers) during a 3-year study period. Seroconversion for Chlamydia pneumoniae was found in 13 of 26 HIV-1 infected children and in 1 of 14 in the seroreverter group (P=0.013). A lower mean CD4+ cell count and p24 antigen positivity at enrolment were significantly associated with seroconversion for Chlamydia pneumoniae. Signs and symptoms of acute respiratory infection were recorded in the 30 to 40 days preceding collection of the blood samples showing seroconversion for Chlamydia pneumoniae in 8 of 13 HIV-1 infected children and in the single seroreverter. This study confirms the potential role of Chlamydia pneumoniae in the pathogenesis of respiratory tract infections in HIV-1 infected subjects.     

Chlamydia pneumoniae Infection Among Healthy Children and Children Hospitalised with Pneumonia in Greece

Chlamydia pneumoniae has been recognized as a cause of respiratory tract infection in humans, and its prevalence has been shown to vary among different age groups and populations. The prevalence of Chlamydia pneumoniae antibody was determined by serological investigation in 343 healthy children and in 77 children consecutively hospitalised for pneumonia in southwestern Greece. Seventy-eight (22.7%) healthy children had IgG Chlamydia pneumoniae titers ≥1/8. The prevalence of Chlamydia pneumoniae antibody in the age groups 6 months–5 years, 6–9 years and 10–15 years was 7.9%, 11.4% and 36%, respectively. One child hospitalised for pneumonia had serological results consistent with acute Chlamydia pneumoniae infection. The results of the present study suggest a low prevalence of Chlamydia pneumoniae antibody among preschoolers in Greece, followed by a steep rise in children 10–15 years of age. Chlamydia pneumoniae is not a common etiologic agent of childhood pneumonia requiring hospitalisation. Electronic Publication     

Phylogeny of Immune Recognition: Processing and Presentation of Structurally Defined Proteins in Channel Catfish Immune Responses

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg^- (T-cell-enriched) and B (sIg⁺) cell subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104.     

Production and Release of Antimicrobial and Immune Defense Proteins by Mammary Epithelial Cells following Streptococcus uberis Infection of Sheep

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.     

Divergent Immune Responses to Mycobacterium avium subsp. paratuberculosis Infection Correlate with Kinome Responses at the Site of Intestinal Infection

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne''s disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/β-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection.     

Recombinant ESAT-6-Like Proteins Provoke Protective Immune Responses against Invasive Staphylococcus aureus Disease in a Murine Model

Staphylococcus aureus is a common pathogen found in the community and in hospitals. Most notably, methicillin-resistant S. aureus is resistant to many antibiotics, which is a growing public health concern. The emergence of drug-resistant strains has prompted the search for alternative treatments, such as immunotherapeutic approaches. To date, most clinical trials of vaccines or of passive immunization against S. aureus have ended in failure. In this study, we investigated two ESAT-6-like proteins secreted by S. aureus, S. aureus EsxA (SaEsxA) and SaEsxB, as possible targets for a vaccine. Mice vaccinated with these purified proteins elicited high titers of anti-SaEsxA and anti-SaEsxB antibodies, but these antibodies could not prevent S. aureus infection. On the other hand, recombinant SaEsxA (rSaEsxA) and rSaEsxB could induce Th1- and Th17-biased immune responses in mice. Mice immunized with rSaEsxA and rSaEsxB had significantly improved survival rates when challenged with S. aureus compared with the controls. These findings indicate that SaEsxA and SaEsxB are two promising Th1 and Th17 candidate antigens which could be developed into multivalent and serotype-independent vaccines against S. aureus infection.     

Analysis of the Immune Responses of Mice to Infection with Leishmania braziliensis

Leishmania major and Leishmania braziliensis both cause cutaneous leishmaniasis, but the former kills BALB/c mice while the latter is killed by the mice. This killing of L. braziliensis occurred by a gamma interferon-dependent mechanism, potentially made possible by the observed lack of high interleukin-4 production.     

Characterization of the Humoral Immune Response to Chlamydia Outer Membrane Protein 2 in Chlamydial Infection

Detection of antibodies to an outer membrane protein 2 (OMP2) by enzyme-linked immunosorbent assay (ELISA) by using either the Chlamydia trachomatis- or the Chlamydia pneumoniae-specific protein was investigated. OMP2 is an immunodominant antigen giving rise to antibody responses in humans infected with different C. trachomatis serovars (A to C and D to K) or with C. pneumoniae, which could be detected by OMP2 ELISA. OMP2 ELISA is not species specific, but antibody titers were usually higher on the homologous protein. The sensitivity of this assay was high but varied according to the “gold standard” applied. Levels of antibody to C. pneumoniae OMP2 as detected by ELISA seem to return to background or near-background values within a shorter period of time compared to antibodies to C. pneumoniae detected by microimmunofluorescence (MIF), making it more likely that positive results in ELISA reflect recent infection. Thus, OMP2 ELISA has distinct advantages over MIF and commercially available ELISAs and might be a useful tool for the serodiagnosis of chlamydial infection.     

Acute and Chronic Phases of Toxoplasma gondii Infection in Mice Modulate the Host Immune Responses

Murine antibody responses to soluble proteins are generally restricted to the immunoglobulin G1 (IgG1) isotype. When mice were infected with Toxoplasma gondii Beverley and concomitantly immunized with a soluble unrelated protein antigen, a modification in the isotypic distribution of antibodies directed against this nonparasite antigen was observed, with a preferential production of IgG2a. Interestingly, when mice were immunized with a soluble protein antigen during the chronic phase (day 40) of infection with T. gondii Beverley, a similar modification in the isotypic distribution of antiprotein antibodies was observed.